Arginine-Functional Methacrylic Block Copolymer Nanoparticles: Synthesis, Characterization, and Adsorption onto a Model Planar Substrate

Recently, we reported the synthesis of a hydrophilic aldehyde-functional methacrylic polymer (Angew. Chem., 2021, 60, 12032–12037). Herein we demonstrate that such polymers can be reacted with arginine in aqueous solution to produce arginine-functional methacrylic polymers without recourse to protecting group chemistry. Careful control of the solution pH is essential to ensure regioselective imine bond formation; subsequent reductive amination leads to a hydrolytically stable amide linkage. This new protocol was used to prepare a series of arginine-functionalized diblock copolymer nanoparticles of varying size via polymerization-induced self-assembly in aqueous media. Adsorption of these cationic nanoparticles onto silica was monitored using a quartz crystal microbalance. Strong electrostatic adsorption occurred at pH 7 (Γ = 14.7 mg m–2), whereas much weaker adsorption occurred at pH 3 (Γ = 1.9 mg m–2). These findings were corroborated by electron microscopy, which indicated a surface coverage of 42% at pH 7 but only 5% at pH 3.


■ INTRODUCTION
Recently, there has been increasing interest in synthetic polymers bearing arginine moieties owing to their potential bioapplications.−7 In view of the development of multidrug-resistant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, 8 there has been a concerted effort to prepare new antimicrobial polymers via reversible addition− fragmentation chain transfer (RAFT) polymerization of arginine-mimicking monomers.For example, Xu et al. grew antimicrobial arginine polymer brushes from planar substrates via surface-initiated RAFT polymerization while Perrier and co-workers designed antibacterial diblock copolymers and antifouling star copolymers using arginine-based acrylamides. 4,9,10Unfortunately, the requirement for Boc or Fmoc protecting groups and the use of toxic organic solvents such as dichloromethane or dioxane significantly reduces the atom economy and cost-effectiveness of many of the above monomer syntheses.In principle, arginine conjugation to aldehyde-functionalized monomers via imine bond formation offers an attractive alternative route to arginine-functionalized polymers.However, until recently, all suitable aldehydefunctional vinyl monomers have been hydrophobic (e.g., 4vinylbenzaldehyde) so their statistical copolymerization with a suitable hydrophilic vinyl monomer has been required to produce the desired water-soluble polymer. 11,12This approach necessarily limits the degree of aldehyde functionality that can be incorporated into such copolymers.
Over the past decade or so, polymerization-induced selfassembly (PISA) has become an established platform technology for the efficient synthesis of a wide range of block copolymer nano-objects.−45 For such syntheses, a water-soluble precursor is first prepared via RAFT solution polymerization and then chain-extended using a suitable vinyl monomer to produce an amphiphilic diblock copolymer.Once a critical degree of polymerization (DP) is achieved, the growing second block becomes insoluble and in situ self-assembly occurs to produce nascent diblock copolymer nanoparticles.−56 Recently, we reported a new synthetic route to controlledstructure poly(amino acid methacrylates). 57First, RAFT solution polymerization of a cis-diol-functional methacrylic monomer GEO5MA using a suitable dithiobenzoate RAFT agent produced a well-defined PGEO5MA homopolymer (M w /M n < 1.20).Subsequently, selective oxidation of the cisdiol groups using NaIO 4 was conducted in aqueous solution to produce an aldehyde-functional water-soluble precursor, followed by (i) reaction with various amino acids (e.g., glycine, lysine, or cysteine) and (ii) reductive amination to afford the desired poly(amino acid methacrylate).This approach was then extended to include various examples of histidinefunctionalized diblock copolymer nano-objects prepared via aqueous PISA 58,59 and polymer brushes 60 prepared via atom transfer radical polymerization. 61erein we exploit the above synthetic strategy to prepare a series of arginine-functionalized diblock copolymer nanoparticles (see Scheme 1).First, GEO5MA is used to prepare a water-soluble PGEO5MA precursor prior to RAFT aqueous dispersion polymerization of benzyl methacrylate to produce sterically stabilized spherical nanoparticles (see Scheme 2).These cis-diol-bearing nanoparticles are then reacted with arginine via Schiff base chemistry, followed by reductive amination using NaCNBH 3 .In principle, arginine addition can occur via the primary amine of the amino acid or via the guanidine moiety to produce a binary mixture of isomers.However, we demonstrate that judicious selection of the solution pH ensures regioselectivity during imine bond formation, thus avoiding the protecting group strategy typically employed by others. 1,2,9,10−65 Accordingly, the physical adsorption of such arginine-functionalized nanoparticles on a model planar substrate is examined using QCM in combination with scanning electron microscopy (SEM).In principle, the arginine-functionalized nanoparticles depicted in Scheme 2 constitute an interesting model system for understanding the pH-modulated adsorption of soft nanoparticles onto a hard planar substrate.
Methods. 1 H NMR Spectroscopy.Spectra were recorded in either D 2 O or d 6 -DMSO using a 400 MHz Bruker Avance-400 spectrometer at 298 K with 16 scans being averaged per spectrum.
Gel Permeation Chromatography.Aqueous gel permeation chromatography (GPC) was used to determine the number-average molecular weights (M n ) and dispersities (M w /M n ) for PGEO5MA 64 , PAGEO5MA 64 , and PArgGEO5MA 64 homopolymers.These polymers were analyzed at 1% w/w using an aqueous buffer eluent containing 0.1 M NaNO 3 , 0.02 M triethylamine, 0.05 M NaHCO 3 , and 0.03% NaN 3 adjusted to pH 10.0 using 1.0 M NaOH.The GPC setup comprised an Agilent 1260 Infinity instrument equipped with a degasser, pump, a guard column, three columns connected in series (PL-Aquagel Mixed-H, OH-30, and OH-40), and a refractive index detector.The column and detector temperature was set at 35 °C, and the flow rate was 1.0 mL min −1 .Calibration was achieved using a series of nine near-monodisperse poly(ethylene oxide) standards ranging from 2.1 to 969 kg mol −1 , and data were analyzed using Agilent GPC/SEC software.
DMF GPC was used to characterize the PGEO5MA 64 homopolymer and all diblock copolymers.These samples were analyzed at 1.0% w/w using HPLC-grade DMF eluent containing 10 mmol LiBr (Figure S1).The GPC setup comprised an Agilent 1260 Infinity instrument equipped with a degasser, pump, a guard column, two PLgel 5 μm Mixed-C columns connected in series, and a refractive index detector.Calibration was achieved using a series of 11 nearmonodisperse poly(methyl methacrylate) standards ranging from 2.38 to 2200 kg mol −1 , and data were analyzed using Agilent GPC/ SEC software.
Dynamic Light Scattering.Dynamic light scattering (DLS) studies were performed using a Malvern Zetasizer Nano-ZS instrument equipped with a 4 mW He−Ne laser (λ = 633 nm) operating at a fixed scattering angle of 173°.Copolymer dispersions were diluted to 0.1% w/w using deionized water prior to light scattering studies at 25 °C, with 2 min being allowed for thermal equilibrium prior to each measurement.The hydrodynamic z-average particle diameter was calculated via the Stokes−Einstein equation, which assumes perfectly monodisperse, noninteracting spheres.The polydispersity index is expressed as a standard deviation that indicates the breadth of the particle size distribution, rather than the experimental error.
Aqueous Electrophoresis.Zeta potentials were determined using a Malvern Zetasizer Nano ZS instrument equipped with a 4 mW He− Ne laser (λ = 633 nm) operating at a fixed scattering angle of 173°.Diblock copolymer nanoparticle dispersions were diluted to 0.1% w/ w with 1 mM KCl as background electrolyte using either dilute HCl or NaOH for pH adjustment as required.Zeta potentials (averaged over three consecutive runs) were calculated via the Henry equation using the Smoluchowski approximation.
Transmission Electron Microscopy.Copper/palladium transmission electron microscopy (TEM) grids (Agar Scientific, UK) were coated in-house to yield a thin film of amorphous carbon and were subjected to a glow discharge for 20 s.An aqueous droplet of a copolymer dispersion (7.0 μL, 0.1% w/w) was placed on freshly treated grids for 1 min and then carefully blotted with filter paper to remove excess solution.An aqueous droplet of uranyl formate solution (5.0 μL, 0.75% w/w) was placed on each sample-loaded grid for 1 min and then blotted with filter paper to remove excess stain.This negative staining protocol was required to ensure sufficient electron contrast.Each grid was then carefully dried using a vacuum hose.Imaging was performed at 80 kV using an FEI Tecnai Spirit 2 microscope fitted with an Orius SC1000B camera.Mean nanoparticle diameters were estimated by digital image analysis using ImageJ software.
QCM Studies.QCM sensors coated with a 50 nm silica overlayer (QSX 303, ̃5 MHz fundamental frequency) were purchased from Biolin Scientific (Gothenburg, Sweden).Each sensor was cleaned according to the manufacturer's instructions.This four-step protocol involved (i) UV/O 3 treatment for 25 min (Bioforce UV/O 3 cleaner, 9 mW cm −2 , λ = 254 nm), (ii) exposure to 2% w/w sodium dodecyl sulfate solution for 30 min, (iii) rinsing with deionized water (iv), drying using a stream of N 2 gas, and (v) a final UV/O 3 treatment for 25 min.QCM measurements were performed using an openQCM NEXT instrument (Novatech Srl., Italy) equipped with a temperature-controlled cell connected to a Masterflex Digital Miniflex peristaltic pump (Cole-Parmer Instrument Co Ltd., St Neots, UK).The cleaned substrates were initially equilibrated with deionized water, then exposed to an aqueous dispersion of 1.0% w/w nanoparticles, and finally washed with deionized water to remove any weakly adhering nanoparticles.Measurements were performed at 25 °C using a flow rate of 0.10 mL min −1 .The mass of adsorbed nanoparticles was calculated using the Sauerbrey equation, which assumes the formation of a rigid thin film and relates the change in resonant frequency (Hz), Δf, to the change in adsorbed mass per unit area, Δm, via a sensitivity constant C (where C = −0.177mg m −2 Hz −1 ) and the harmonic number n.
−68 Scanning Electron Microscopy.After nanoparticle adsorption experiments, selected silica-coated QCM sensors were sputter-coated with a 5 nm layer of gold and SEM images were captured using an Inspect-F instrument operating at an accelerating voltage of 10 kV and a beam current of 200 nA.
Synthetic Protocols.Synthesis of the PGEO5MA 64 Precursor via RAFT Solution Polymerization in Ethanol.GEO5MA monomer (30.0 g, 0.079 mol), CPDB (0.146 g, 0.66 mmol), ACVA initiator (0.037 g, 0.132 mmol; CPDB/ACVA molar ratio = 5.0), and ethanol (20.0 g) were weighed into a 50-mL round-bottomed flask.This reaction mixture was degassed with N 2 for 30 min, and the flask was placed in an oil bath set at 70 °C for 120 min.The polymerization was quenched by removing the flask from the oil bath and exposing its contents to air while cooling to 20 °C. 1 H NMR studies confirmed the GEO5MA conversion to be 53% as judged by the attenuation of the GEO5MA vinyl protons at 5.7−6.1 ppm to the overlapping PGEO5MA and GEO5MA monomer oxymethylene proton signals at 4.1 and 4.3 ppm, respectively.Crude PGEO5MA was purified by precipitation into excess diethyl ether.The precipitate was redissolved in methanol, and the precipitation step was repeated.The PGEO5MA product was redissolved in deionized water, dialyzed for 2 days (with three changes of water per day), and then freeze-dried overnight to produce a red viscous liquid.The mean DP of the purified PGEO5MA precursor was estimated to be 64 via end-group analysis using 1 H NMR spectroscopy (the integrated aromatic protons assigned to the phenyl end-group derived from the RAFT agent at 7.3−8.0ppm were compared to the integrated methacrylic backbone protons at 0.80−2.30ppm).
Oxidation of PGEO5MA 64 Homopolymer Using NaIO 4 .PGEO5MA 64 homopolymer (0.30 g, 12.2 μmol) and 0.70 g of deionized water were weighed into a 15-mL vial and stirred to produce an aqueous solution.Then an aqueous solution of 0.39 M NaIO 4 (2.0 mL) was added, and the reaction mixture was stirred for 5 min at 25 °C.A NaIO 4 /GEO5MA molar ratio of unity was targeted to ensure full oxidation of the pendent cis-diol groups.The degree of oxidation was estimated using 1 H NMR spectroscopy by comparing the integrated proton signal adjacent to the geminal diol group at 5.1 ppm to that of the oxymethylene proton signal at 4.1 ppm.The resulting 10% w/w aqueous solution of PAGEO5MA was dialyzed against deionized water for two days (with three changes of water per day).
Reductive Amination of PAGEO5MA 64 Homopolymer Using Arginine and NaCNBH 3 .L-Arginine (49.5 mg, 0.285 mmol) was dissolved in a 10% w/w aqueous solution of PAGEO5MA 64 homopolymer (1.00 g), and the resulting solution was adjusted to either pH 6 (using 0.1 M HCl) or pH 10 (using 0.1 M NaOH).Then NaCNBH 3 (43.8mg, 0.698 mmol) was added, and the reaction mixture was stirred at 35 °C for 15 min before being dialyzed against deionized water for 2 days (with three changes of water per day) to remove impurities and any unreacted reagents.The degree of arginine functionalization was estimated by using 1 H NMR spectroscopy to monitor the disappearance of the geminal diol proton signal at 5.1 ppm relative to the methacrylic backbone proton signals at 0.8−2.3ppm.The selectivity of this derivatization was estimated by comparing the −CH 2 −CH 2 −NH− signal intensity at 3.1 ppm from the two azamethylene protons associated with the amino acid group (see signal c in Figure 2) to that at 3.2 ppm from the two aza-methylene protons associated with the guanidine group (see signal d in Figure 2).

Synthesis of PGEO5MA 64 −PBzMA x Nanoparticles via RAFT Aqueous Emulsion Polymerization of Benzyl Methacrylate.
The following synthesis of PGEO5MA 64 −PBzMA 500 nanoparticles at 16.6% w/w solids is representative of the general protocol.BzMA monomer (0.31 g, 1.76 mmol), PGEO5MA 64 precursor (0.086 g, 3.5 μmol; target PBzMA DP = 500), ACVA initiator (0.30 mg, 1.2 μmol; PGEO5MA 64 /ACVA molar ratio = 5.0), and water (2.0 g; targeting 16.6% w/w solids) were weighed into a 15 mL glass vial.The mixture was purged with N 2 for 15 min, and the vial was placed in an oil bath at 70 °C.After 16 h, the BzMA polymerization was quenched by removing the vial from the bath and exposing the resulting aqueous dispersion to air while cooling to 20 °C.The final BzMA conversion was determined to be more than 99% via 1 H NMR spectroscopy by monitoring the reduction in intensity of the vinyl proton signals at 5.6−6.2ppm relative to that of the methacrylic backbone signals at 0.80−2.30ppm (Figure S2).
Selective Oxidation of PGEO5MA 64 −PBzMA 500 Nanoparticles Using NaIO 4 .Oxidation of PGEO5MA 64 −PBzMA 500 nanoparticles at 8.3% w/w solids was conducted by adding 1.00 g of an aqueous solution of 0.10 M NaIO 4 to a 15 mL glass vial containing 1.00 g of a 16.6% w/w aqueous dispersion of PGEO5MA 64 − PBzMA 500 nanoparticles (0.166 g, 1.47 μmol); the resulting reaction mixture was stirred for 5 min at 25 °C.A NaIO 4 /GEO5MA molar ratio of unity was employed to target full oxidation of the pendent cisdiol groups.The mean degree of oxidation was estimated using 1 H NMR spectroscopy in d 6 -DMSO (Figure S3).The resulting 8.3% w/w aqueous dispersion of PAGEO5MA 64 −PBzMA 500 nanoparticles was dialyzed against deionized water for 2 days (with three changes of water per day).

■ RESULTS AND DISCUSSION
Synthesis of an Arginine-Functionalized Water-Soluble Homopolymer.We have recently reported the synthesis of the PGEO5MA precursor and its corresponding hydrophilic aldehyde-functional polymer (PAGEO5MA 64 ), elsewhere. 57In principle, PAGEO5MA 64 can be derivatized with various amine-functionalized molecules (e.g., amino acids, oligopeptides, proteins or dyes). 57−59 For example, using arginine should yield an arginine-functionalized polymer, PArgGEO5MA.However, in our initial experiments, we found that reductive amination of PAGEO5MA using arginine at pH 6 yielded a binary mixture of isomers owing to a lack of regioselectivity under such conditions.More specifically, the desired major isomer (arginine attached via the N-terminus, see Figure 1) comprised only 79% of the isomeric mixture.This problem arises because both the primary amine and guanidine groups in arginine are protonated at pH 6.Hence, there is insufficient difference between these two potential reactive sites to ensure selectivity.
Fortunately, the primary amine group within arginine (pK a 9.0) exists mainly in its neutral (nonprotonated) form at pH 10, while the guanidine group (pK a 13.8) should remain in its

Biomacromolecules
protonated form under such conditions. 69In principle, this should be sufficient to achieve the desired selectivity.Accordingly, the reductive amination of PAGEO5MA 64 using arginine was performed at pH 6 and pH 10, and the product(s) of these reactions were analyzed by 1 H NMR spectroscopy (Figure 2).Periodate oxidation of the PGEO5MA 64 precursor to form PAGEO5MA 64 produced two new proton signals associated with the geminal diol group, which is the hydrated form of aldehyde that is obtained in water (Figure 2b).Importantly, reductive amination of PAGEO5MA 64 with arginine at pH 10 yielded a single product (Figure 2c), as opposed to the binary mixture of isomers obtained at pH 6 (Figure 2d).Clearly, reductive amination of PAGEO5MA 64 with arginine at pH 10 provides a highly convenient wholly aqueous route to well-defined arginine-functionalized polymers.
Aqueous GPC was used to characterize the PGEO5MA 64 precursor, the aldehyde-functionalized PAGEO5MA 64 , and the arginine-functionalized PArgGEO5MA 64 (Figure 3).Oxidation of PGEO5MA 64 to PAGEO5MA 64 involves the loss of formaldehyde, which results in a discernible reduction in M n from 9.9 to 8.1 kg mol −1 .As expected, functionalization of PAGEO5MA 64 with arginine results in a significantly higher M n for PArgGEO5MA 64 .Moreover, the molecular weight distributions obtained for all three polymers are relatively narrow (M w /M n = 1.21−1.25),which indicates that each homopolymer is well-defined and that no side-reactions (e.g., branching or cross-linking) occurred during either oxidation or reductive amination.
Synthesis and Characterization of Arginine-Functionalized Diblock Copolymer Nanoparticles.Chain extension of this water-soluble dithiobenzoate-capped PGEO5MA 64 precursor via RAFT aqueous emulsion polymerization of benzyl methacrylate (BzMA) at 70 °C produced a series of PGEO5MA 64 −PBzMA x nanoparticles.DMF GPC studies confirmed efficient chain extension and a relatively narrow molecular weight distribution in each case (Figure S1).Systematic variation of the target DP for the core-forming PBzMA x block from 50 to 500 produced six aqueous nanoparticle dispersions, with DLS studies indicating z-average diameters ranging from 31 to 61 nm (Figure 4a).A plot of such data reveals a monotonic increase in nanoparticle diameter (Figure S4).Similarly, the corresponding TEM images suggest a monotonic increase in the number-average diameter of the nanoparticle cores, D n , in accordance with prior aqueous PISA formulations (Figure 4b−g). 59,70,71In addition, the mean aggregation number, N agg , or average number of copolymer chains per nanoparticle, was estimated for the smallest and largest nanoparticles.More specifically, the PBzMA core volume, V, was calculated from D n using V = 1 6 πD n 3 .Hence the corresponding PBzMA core mass, m, is calculated using m = ρ•V, where the density of PBzMA, ρ, is 1.15 g cm −3 ; dividing m by the molar mass of the PBzMA x chains gives the mean aggregation number N agg .Hence PGEO5MA 64 −PBzMA 50 nanoparticles have an N agg of 113, while the PGEO5MA 64 −PBzMA 500 nanoparticles have an N agg of 177.
The largest PGEO5MA 64 −PBzMA 500 nanoparticles were selected for subsequent derivatization to aid their visualization after adsorption.Accordingly, NaIO 4 oxidation yielded the corresponding aldehyde-functional PAGEO5MA 64 −PBzMA 500 nanoparticles, which were subsequently derivatized with arginine via reductive amination at pH 10 to yield cationic PArgGEO5MA 64 −PBzMA 500 nanoparticles.DLS and TEM  studies of the corresponding nanoparticles confirmed that their morphology was not adversely affected during each derivatization (see Figure 5).
Aqueous electrophoresis was employed to assess the change in electrophoretic behavior of these nanoparticles during their derivatization.Accordingly, zeta potential versus pH curves were constructed from pH 2 to 10.As expected, the cis-diolfunctionalized PGEO5MA 64 −PBzMA 500 precursor nanoparticles and the aldehyde-functional PGEO5MA 64 −PBzMA 500 nanoparticles remained essentially neutral across the whole pH range (see Figure 6a and 6b, respectively).In contrast, the PArgGEO5MA 64 −PBzMA 500 nanoparticles exhibit significant cationic character.A zeta potential of around +34 mV is observed at pH 2, which corresponds to the regime in which the pendent primary amine and guanidine groups are both protonated, and the pendent carboxylic acid group is in its neutral (non-ionized) form.A gradual reduction to a plateau value of +22 mV occurs on raising the pH to 4.3, which then remains constant up to pH 7.2.In this second regime, the carboxylic acid group becomes ionized, which lowers the overall cationic surface charge.A further gradual reduction in zeta potential occurs thereafter owing to deprotonation of the pendent primary amine group, with essentially neutral character observed for these nanoparticles at around pH 10 (Figure 6c).Essentially the same zeta potential versus pH curve was obtained for the smaller PArgGEO5MA 64 −PBzMA 50 nanoparticles (Figure S5).
Adsorption Studies of Arginine-Functionalized Diblock Copolymer Nanoparticles.Adsorption of the cationic PArgGEO5MA 64 −PBzMA 500 nanoparticles onto a model planar substrate (silica) was studied at pH 7 using a QCM (Figure 7a).In such experiments, adsorbed nanoparticles are considered to form a rigid thin film so the Sauerbrey equation is valid.Strong nanoparticle adsorption (Γ = 14.7 mg m −2 ; red curve) is observed at 25 °C.The silica surface is highly anionic at pH 7, which leads to electrostatic adsorption of the cationic nanoparticles.In contrast, despite their greater cationic character (see Figure 6c), nanoparticle adsorption is substantially reduced at pH 3 (Γ = 1.9 mg m −2 ; orange curve).This is because the silica substrate exhibits almost no surface charge under these conditions so nanoparticle adsorption involves only van der Waals interactions.In a control experiment, the neutral PGEO5MA 64 −PBzMA 500 precursor nanoparticles were also adsorbed onto silica at pH 7. In this case, similarly weak adsorption (Γ = 2.3 mg m −2 ; black curve) was observed, again owing to the absence of any electrostatic attractive interactions.In both cases, the silica sensor was rinsed with deionized water immediately after nanoparticle adsorption, but no discernible change in frequency was observed.
SEM images were recorded for the QCM sensors after performing adsorption experiments using PArgGEO5MA 64 − PBzMA 500 nanoparticles (Figure 7b).Using digital image analysis (ImageJ software), a surface coverage of 42% was calculated for electrostatic adsorption at pH 7 but just 5% surface coverage was estimated for the same nanoparticles adsorbed at pH 3. In summary, these experiments confirm that the extent of adsorption of arginine-functionalized PArgGEO5-MA 64 −PBzMA 500 nanoparticles onto silica is strongly pHdependent.
Finally, QCM was used to study the adsorption of the smallest [DLS diameter = 31 nm (0.14)] PArgGEO5MA 64 − PBzMA 50 nanoparticles at pH 7 (Figure 8).As expected, an appreciably lower adsorbed amount (Γ = 10.6 mg m −2 ) was observed compared to that obtained for the 61 nm DLS diameter PArgGEO5MA 64 −PBzMA 500 nanoparticles (compare green and red curves).Brotherton et al. reported similar observations for the adsorption of sterically stabilized nanoparticles onto stainless steel from aqueous solution. 59Moreover, the relatively small PArgGEO5MA 64 −PBzMA 50 nanoparticles are clearly less strongly adsorbed at the silica surface because a minor fraction (8%) could be removed when rinsing with deionized water.In contrast, no reduction in the adsorbed amount occurs for the larger PArgGEO5MA 64 −PBzMA 500 nanoparticles.Similar observations were made for the neutral PGEO5MA 64 −PBzMA 50 and PGEO5MA 64 −PBzMA 500 nano-particles: a substantial proportion (68%) of the former could be removed by rinsing, whereas the adsorbed amount obtained for the latter remained essentially unchanged after rinsing (compare blue and black curves).Thus smaller nanoparticles adhere more weakly than larger nanoparticles in the absence of a strong electrostatic attractive interaction between the nanoparticles and the planar substrate.Conversely, introducing such an electrostatic interaction can minimize the partial loss of relatively small nanoparticles during rinsing.In summary, this is an interesting new model system for understanding the effect of particle size and electrostatic attractive forces on the (ir)reversible adsorption of electrosterically stabilized nanoparticles onto oppositely charged planar surfaces.

■ CONCLUSIONS
We demonstrate that a hydrophilic aldehyde-functional methacrylic polymer can be reacted with arginine in aqueous solution under mild conditions to produce the analogous arginine-functional methacrylic polymer.Importantly, this chemical derivatization can be achieved without recourse to protecting group chemistry.Careful control of the solution pH is essential to ensure regioselectivity for initial imine bond formation; subsequent reductive amination using NaCNBH 3 leads to a hydrolytically stable amide linkage.This protocol was then utilized to prepare arginine-functionalized diblock copolymer nanoparticles in aqueous media via PISA.Such functionalization did not adversely affect either the nanoparticle size distribution or the molecular weight distribution of the derivatized diblock copolymer chains.Aqueous electrophoresis studies confirmed that these arginine-functionalized nanoparticles exhibit cationic character between pH 2 and 9.A QCM instrument was used to study the adsorption of the resulting cationic nanoparticles onto a planar silica surface.Favorable electrostatic interactions led to strong adsorption at pH 7 (Γ = 14.7 mg m −2 ).In contrast, much weaker adsorption was observed at pH 3 (Γ = 1.9 mg m −2 ) because the silica substrate has almost no anionic surface charge under such conditions.These findings were corroborated by SEM studies, which indicated surface coverages of 42% at pH 7 and 5% at pH 3, respectively.Finally, minimal nanoparticle adsorption was also observed at pH 10 because the nanoparticles are close to their isoelectric point under such conditions.■ ASSOCIATED CONTENT * sı Supporting Information

Scheme 1 .
Scheme 1. Synthesis of PGEO5MA 64 via RAFT Solution Polymerization of GEO5MA, Followed by Its Selective Oxidation Using NaIO 4 in Aqueous Media to Produce the Corresponding Aldehyde-Functional Polymer (PAGEO5MA 64 ) a

Scheme 2 .
Scheme 2. Synthesis of PGEO5MA 64 −PBzMA x Nanoparticles via RAFT Aqueous Emulsion Polymerization of Benzyl Methacrylate at 70°C a

Figure 1 .
Figure 1.Schematic cartoon depicting the two possible isomers that may be formed when reacting PAGEO5MA 64 with arginine in the presence of NaCNBH 3 .

Figure 2 .
Figure 2. Effect of solution pH on regioselectivity.Partial 1 H NMR spectra recorded in D 2 O (pH 6; using solvent suppression) for each step during the synthesis of PArgGEO5MA 64 : (a) cis-diol functional PGEO5MA 64 precursor; (b) aldehyde-functional PAGEO5MA 64 ; (c) PArgGEO5MA 64 produced via reductive amination with arginine at pH 10 (regioselectivity under such conditions yields a single isomer); (d) binary mixture of PArgGEO5MA 64 products obtained via reductive amination with arginine at pH 6 (in this case, poor regioselective control produces two isomers).

Figure 3 .
Figure 3. Aqueous GPC curves recorded for the PGEO5MA 64 precursor prepared via RAFT aqueous solution polymerization of GEO5MA (black curve), the aldehyde-functional PAGEO5MA 64 homopolymer prepared via selective oxidation of this PGEO5MA 64 precursor (purple curve), and the PArgGEO5MA 64 homopolymer obtained via reductive amination after the Schiff base reaction of PAGEO5MA 64 with arginine at pH 10 (green trace).Apparent M n values are expressed relative to a series of near-monodisperse poly(ethylene oxide) calibration standards.

Figure 4 .
Figure 4. Particle size control by systematic variation of target PBzMA DP.(a) DLS particle size distributions (including z-average diameters and DLS polydispersities) and (b−g) corresponding TEM images recorded for a series of six examples of PGEO5MA 64 − PBzMA x nanoparticles prepared via RAFT aqueous emulsion polymerization of benzyl methacrylate at 70 °C when targeting a PBzMA DP of 50−500.

Figure 6 .
Figure 6.Aqueous electrophoresis data.Zeta potential versus pH curves obtained in the presence of 1 mM KCl for: (a) cis-diolfunctionalized PGEO5MA 64 −PBzMA 500 nanoparticles; (b) aldehydefunctionalized PAGEO5MA 64 −PBzMA 500 nanoparticles; (c) argininefunctionalized PArgGEO5MA 64 −PBzMA 500 nanoparticles.The two vertical dashed lines at pH 4.2 and pH 7.2 correspond to the approximate pK a values for the deprotonation of the carboxylic acid and the primary amine of the pendent amino acid group in PArgGEO5MA 64 , respectively.

Figure 8 .
Figure 8.Effect of particle size and chemical functionality on nanoparticle adsorption at a planar silica substrate.QCM curves recorded at pH 7 during adsorption of the smallest (DLS diameter = 31 nm, blue trace) and largest (DLS diameter = 61 nm, black trace) cis-diol-functionalized PGEO5MA 64 −PBzMA x nanoparticles and the corresponding cationic arginine-functionalized PArgGEO5MA 64 − PBzMA x nanoparticles (red and green traces, respectively).The four asterisks indicate the points at which deionized water was introduced to remove any weakly adhering nanoparticles.